Background and Aims: We demonstrated that physical training, characterized by repeated ischemia-reperfusion (I-R) episodes (ischemic conditioning, IC), protects circulating cells from peripheral artery disease (PAD) patients against ischemic harms by reducing oxidative stress (OS) and by up-regulating nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway expression. Ezetimibe (Eze) has been shown to alleviate OS enhancing Nrf2 nuclear translocation in an AMPK/p62-dependent manner. In a cellular I-R and IC model, we aimed to investigate: 1) the effect of Eze on OS and Nrf2/ARE gene expression 2) whether Eze could have a synergistic effect on IC. Methods: THP-1 cells were treated with or without Eze (50mM) overnight, then subjected to 1 or 6 repetitive I-R cycles using EVOS FL Auto Imaging System. Reactive oxygen species (ROS) formation was evaluated with DCF in cytofluorimetry. Nrf2/ARE and p62 gene expression were evaluated by RT-PCR and western blotting. Results: When THP-1 cells were exposed to 1 I-R cycle, the preincubation with Eze significantly reduced ROS formation (p<0.01) and up-regulated Nrf2/ARE pathway expression and p62 phosphorylation (p<0.001). Multiple I-R cycles, acting as IC, significantly reduced ROS formation and upregulated Nrf2/ARE gene expression (p<0.001); in these conditions, Eze preincubation was able not only to almost abolish ROS formation (p<0.01) but also further up-regulate Nrf2/ARE expression. Conclusions: In our I-R model, Eze not only restores I-R-induced oxidative damages through Nrf2/ARE signaling up-regulation but also has a synergistic effect on IC. This new \u201cpleiotropic\u201d effect, if confirmed in vivo, may strengthen the use of Eze in PAD patien
